Method of calibrating and correcting color-bleed factors for color separation in dna analysis

ABSTRACT

A method includes calibrating color bleed factors of optical detector channels of a sample processing apparatus through processing a color bleed calibration substance which includes a plurality of different size fragments replicated from different groups of DNA loci, wherein fragments in a same group are labeled with a same fluorescent dye, and fragments in different groups are labeled with different fluorescent dyes having different emission spectra, wherein the different size fragments are processed during different acquisition times.

TECHNICAL FIELD

The following generally relates to DNA analysis and finds particularapplication to color bleed factor calibration for color separation inDNA analysis. However, the following is also amenable to otherapplications.

BACKGROUND

DNA genotyping is a process of determining the sequence of DNAnucleotides at a generic locus, or at a position on a chromosome of agene or other chromosome marker. For the purpose of identifying a human,certain generic loci have been selected as the standard markers tocharacterize the DNA. Each marker is a DNA fragment containing arepetition of a certain nucleotide sequence. Generally, there are 13cores and several other accepted standard markers by the securityauthorities. These markers contain short repetitions (e.g., roughly from5 to 40) of four nucleotides. They are in the class of Short TandemRepeat (STR) of DNA sequence.

The repetition numbers at these markers varies rather randomly fromperson to person. The specific form of DNA sequence at a generic locusis called an allele, which provides sufficient differentiation amongpeople. The STR sequence is inherited from parent's DNA. At each marker,there may be two different alleles, one from each parent, and it iscalled heterozygous. If the alleles from both parents have same STRnumbers, it is homozygous. If the alleles of 13 core markers wereheterozygous, each person will have 26 different allele numbers. Assumeeach number is evenly distributed over a range of 10, the likelihood ofhaving two people with the same alleles numbers from these 13 markers isextremely small.

To measure allele numbers, a DNA fragment containing all STR nucleotidesand adjacent sections of nucleotides at each locus is copied from theDNA sample, and replicated by a technique called polymerase chainreaction (PCR). The fragment size is measured in the unit of base pairs,where a base pair is the size of a pair of DNA nucleotides. The sampleis placed in a capillary of a sample carrier, and the fragments areseparate by size through electrophoresis in which same size fragmentsarrive at a destination at about the same time, and different sizefragments arrive at the destination at different times.

A modern apparatus for DNA analysis uses a rigid sample carrier calledbiochip which contains multiple capillaries in parallel to run multiplesamples simultaneously. To detect the fragments, a fluorescent dye isattached to the fragments and the sample is excited by a light source ofnarrow beam at a fixed spot of the capillary. The fluorescent dye isalso called fluorophore, and its attachment to fragments is also said tolabel the fragments. Following the excitation, fluorescent light isemitted from the dye very much instantaneously, typically within onemicrosecond.

The sizes of the fragments in a DNA locus are known to be within certainrange. It is possible to find a number of loci in which the fragmentsizes of a locus do not overlapped with other loci. Furthermore, it ispossible to divide the whole set of loci into several groups. In eachgroup, the fragment sizes of a locus are separated from other loci, andit is called a color group. The fragment size is measure in DNA basepairs and it is ranged from 100 to 400 base pairs in the figure. Foreach color group, a dye with a distinct fluorescent color is attached tothe fragments of all loci in the group. Usually, the dye is attached toa molecule called primer at one end of the fragment. The fragments areseparated by the electrophoresis process and detected by an opticalsystem as a digital signal. A fragment is detected as a peak in thesignal, and the detection time of a peak can be used to determine thefragment size.

Based on the non-overlapping range of the loci in the color group, themeasured fragment size identifies the locus of the fragment. With othersupporting data, the measured fragment size can be used to identify itas one of DNA fragments in the locus with known STR number. The sampleis prepared with multiple dyes with one dye for each color group. Whenthe sample is excited by the light source, the fluorescent light ismixed with multiple colors from these dyes. It is necessary to useoptical filter to separate the fluorescent colors. Each filteredfluorescent color is measured in a detection channel as an electricalsignal. Typically, a photo-multiplier tube (PMT) or other detectors,such as charge-coupled device (CCD) camera is used in each detectionchannel.

Ideally, the emission spectrum of each dye is narrow such that thespectra of the multiple dyes in the sample do not overlap each other. Ifthat were the case and if the optical filter could also be narrow bandto detect only one dye, then each of the detected signals would containonly one dye color. In this hypothetic ideal case, each signal measuresone and only one color group, in which a DNA fragment peak would onlyappear in one of the detected signals. By finding and identifying thepeaks in these signals, the complete set of STR numbers in all loci ofinterest can be determined.

However, the emission spectra of the dyes overlap with each othersubstantially. As the result, each detected signal contains fluorescentsignals from all dyes. This has been referred to as color-bleed, and issimilar to the cross-talk problems in electronic instruments. Withconventional systems, the degrees of color-bleed can be severe, and itis necessary to know the degree of color-bleed from each dye as accurateas possible. The degree is used as a set color-bleed factors that areused to determine signals corresponding to only one distinct color froma dye through a process referred to as color separation. Unfortunately,an inaccurate set of color-bleed factors can lead to false peaks and/oramplitude-diminished true peaks, which may lead to uncertainty withdetermining STR numbers.

SUMMARY

Aspects of the application address the above matters, and others.

In one aspect, a method includes calibrating color bleed factors ofoptical detector channels of a sample processing apparatus throughprocessing a color bleed calibration substance which includes aplurality of different size fragments replicated from different groupsof DNA loci, wherein fragments in a same group are labeled with a samefluorescent dye, and fragments in different groups are labeled withdifferent fluorescent dyes having different emission spectra, whereinthe different size fragments are processed during different acquisitiontimes.

In another aspect, a method includes generating a first signalindicative of a reference gain of optical detectors of a sampleprocessing apparatus based on a first emission from a gain-monitoringmaterial of the sample processing apparatus in response to illuminatingthe material, generating a second signal indicative of a subsequent gainof the optical detectors based on a second emission from again-monitoring material in response to illuminating the material, andscaling at least one of color bleed factors of the sample processingapparatus or data acquired by the sample processing apparatus based on asignal indicative of a difference between the reference gain and thesubsequent gain.

In another aspect, a sample processing system includes a sample carrierreceptacle configured to receive a sample carrier carrying one or moresamples to be processed by the sample processing system. The sampleprocessing system further includes one or more processing stations forprocessing the one or more samples. The sample processing system furtherincludes a reader, including an illumination source and one or moreoptical detector channels, that evaluates separated fragments of aprocessed sample based on emission spectrums of dyes attached to thefragments, and that generates an output signal. The sample processingsystem further includes a color separator that color separates a readeroutput signal corresponding to a processed DNA sample based on colorbleed factors of the one or more optical detector channels. The sampleprocessing system further includes a color bleed factor generator and/orcorrector configured to determine color bleed factors for the opticaldetector channels based on processing a color bleed calibrationsubstance, wherein the color bleed calibration substance includes aplurality of different size fragments in which different size fragmentsare grouped and labeled with different dye having different emissionspectrums in different groups, and the different size fragments areprocessed and detected over different acquisition times.

In another aspect, a sample processing system includes a sample carrierreceptacle configured to receive a sample carrier carrying one or moresamples to be processed by the sample processing system. The systemfurther includes one or more processing stations for processing the oneor more samples. The system further includes a gain-monitoring devicethat emits light or a gain-monitoring material that emits fluorescentlight with a wide spectrum. The system further includes a reader,including an illumination source and one or more optical detectorchannels, that evaluates the gain-monitoring material and separatedfragments of a processed sample based on emission spectrums of dyesattached to the fragments, and that generates an output signal. Thesystem further includes a color separator that color separates an outputsignal of the reader corresponding to a processed DNA sample based oncolor bleed factors of the one or more optical detector channels. Thesystem further includes a color bleed factor generator and/or correctorconfigured to determine a gain of the detector channels based on thesignal emitted by the gain-monitoring material and correct the colorbleed factors for changes in gain of the optical detector channels.

In another aspect, a color bleed calibration substance includes aplurality of different size DNA fragments in which fragments of the samelocus are prepared and labeled with the same fluorescent dye and thedifferent size fragments are processed during different acquisitiontimes by a sample processing system, wherein emission of the fluorescentdyes in response to being illuminated provides a signal in indicative ofcolor bleed factors of detection channels of an optical reader of thesample processing system.

Those skilled in the art will recognize still other aspects of thepresent application upon reading and understanding the attacheddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The application is illustrated by way of example and not limitation inthe figures of the accompanying drawings, in which like referencesindicate similar elements and in which:

FIG. 1 illustrates an example sample processing apparatus;

FIG. 2 illustrates an example sub-portion of a distribution of a colorbleed calibration substance;

FIG. 3 illustrates an example of color bleed of a dye across opticaldetectors;

FIG. 4 illustrates an example color bleed factor determiner fordetermining and/or correcting color bleed factors based on a calibrationsubstance;

FIG. 5 illustrates an example of determining peak areas for determiningcolor bleed factors;

FIG. 6 illustrates an example of the one or more gain-monitoringfixtures in connection with the sample processing apparatus;

FIG. 7 illustrates an example color bleed factor determiner forcorrecting color bleed factors based on emissions from one or moregain-monitoring materials or light source;

FIG. 8 illustrates an example method for determining and/or correctingcolor-bleed factors based on a calibration substance; and

FIG. 9 illustrates an example method for correcting color-bleed factorsbased on emission from one or more gain-monitoring materials or lightsource.

DETAILED DESCRIPTION

FIG. 1 illustrates a sample processing apparatus 102.

The illustrated apparatus 102 is configured for processing one or moresamples carried by a sample carrier 104. A suitable sample carrier 104includes, but is not limited to, a biochip, a lab-on-a-chip, and/orother sample carrier. Such a sample carrier 104 may include one or moremicro-channels for carrying and moving, in parallel and/or in series,one or more samples through a plurality of different processing regionsof the sample carrier 104. Suitable samples include, but are not limitedto, a bio-sample (e.g., saliva, blood, skin cells, and/or otherbio-material), a non-bio sample, etc. The sample processing apparatus102 includes a sample carrier receptacle 106 configured to receive thesample carrier 104.

The sample processing apparatus 102 further includes one or moreprocessing stations 108 ₁, . . . , 108 _(N) (wherein N is an integerequal to or greater than one), collectively referred to herein asprocessing stations 108. The illustrated sample processing apparatus 102is configured to process samples carried by the sample carrier 104received by the sample carrier receptacle 106. In one instance, suchprocessing includes processing DNA samples carried by the sample carrier104. In this instance, the processing stations 108 are configured tosuch functions as extract and purify DNA fragments, replicate and labelthe DNA fragments with fluorescent dyes having known emission spectrums(or colors), separate the labeled fragments based on fragment size, forexample, via electrophoresis, and detect the fragments based on theemission spectrum of the dyes.

The sample processing apparatus 102 also includes an optical reader 110.The reader 110 includes a light source that directs a light beam of apredetermined wavelength range at the separated fragments. In oneinstance, the light source emits a relatively narrow light beam with adiameter in the order of 10 to 100 microns. In another instance, thelight source emits a light beam with a smaller or large diameter.Examples of suitable light sources include, but are not limited to, alaser, a light emitting diode (LED), and the like. The reader 110 alsoincludes an optical detection channel (e.g., a photo-multiplier tube(PMT), a charge-coupled device (CCD) camera, or the like) for eachwavelength range (or color) of interest that generates an electricalsignal in proportion to the intensity of the fluorescence light withinthe wavelength range.

A color separator 112 color-separates the signals from the reader 110based on a set of color bleed factors. Generally, the emission spectraof the dyes attached to the fragments overlap. As the result, the outputsignal of a detection channel not only will include peaks correspondingto the wavelength of interest of the detection channel, but alsopossibly peaks from wavelengths of the one or more of the otherdetection channels. The set of color-bleed factors describes the degreeof color-bleed and is used to correct the signals so that each signalmeasures only one distinct color from a dye. A STR determiner 114identifies the peaks in the signals and determines STR (Short TandemRepeat) numbers in loci of interest based on the identified peaks.

A color bleed factor determiner and/or corrector 116 can be used togenerate an initial set of color bleed factors and/or, subsequently, acorrection thereto before, during and/or after processing a sample. Inone instance, the correction compensates for changes in detector channelgain over time. As described greater detail below, the color bleedfactors can be determined based on processing a color bleed substance,and the correction thereto can be determined based on processing thecolor bleed substance, a positive control sample having characteristicsof the color bleed substance, and/or one or more gain-monitoringfixtures 118.

A signal router 120 routes the output of the reader 110 to the colorseparator 112 and/or the color-bleed factor generator and/or corrector116 based on a mode of operation. A controller 122 generates a signalindicative of a selected mode of operation and conveys the signal to thesignal router 120, and the router 120 routes the signal based on thesignal. A user interface 124 allows a user of the apparatus to selectthe mode of operation. Examples of modes include a DNA processing mode,a calibration mode, such as a pre run time, run time, and/or post runtime calibration mode, and/or one or more other modes.

It is to be appreciated that the sample processing apparatus 102 may beconfigured to be a portable apparatus that can be readily moved fromlocation to location. In another embodiment, the sample processingapparatus 102 is configured to be a stationary apparatus mounted to orplaced on a table, the floor, etc. in a laboratory, office, or the likeand configured to remain at a particular location.

As briefly discussed above, the color bleed factor determiner and/orcorrector 116 can be used to determine a set of color-bleed factors. Inone instance, the color bleed factor determiner and/or corrector 116determines the set of color bleed factors based on processing acalibration substance. This is illustrated through FIGS. 2, 3, 4 and 5.

FIG. 2 shows a sub-portion of a color bleed calibration substance as afunction of dye (color) and fragment size with five different dyes, eachhaving a different emission spectrum range. A y-axis 202 represents thedyes and an x-axis 204 represents fragment size, and fragments 206 and208, 210 and 212, 214 and 216, 218 and 220, and 222 and 224 respectivelycorrespond to dyes 226, 228, 230, 232, and 234. The different sizefragments are replicated from DNA loci of interest, and fragments in asame group are labeled with a same fluorescent dye, and fragments indifferent groups are labeled with different fluorescent dyes. Note thatthe fragments for the different dyes do not overlap in fragment size.

In another example, the color bleed calibration substance includes lessthan five dyes, and multiple substances are concurrently utilized. Inyet another example, a positive control sample with known fragmentssizes can be used as the color bleed substance if the positive controlsample does not include same size fragments labeled with different dyes.It is to be appreciated that the color bleed calibration substance canalso be used as a positive control sample.

FIG. 3 shows the output of five detection channels of the reader 110(FIG. 1) for the fragment size 214 attached with dye 230 of thecalibration substance of FIG. 2. A y-axis 302 represents detectorchannel output and an x-axis 304 represents the acquisition time inwhich the fragment 214 is processed. In this example, peak 306represents the output 316 of detector channel 326, which is the detectorchannel configured to detect the emission of the dye 230 attached to thefragment size 214. Peaks 308, 310, 312 and 314 represent the outputs318, 320, 322 and 326 of the other detector channels 328, 330, 332 and334, which detect fractional amounts of the emission of the dye 230.

FIG. 4 illustrates an example color bleed factor determiner and/orcorrector 116 used to determine a set of color-bleed factor based on theoutput peaks 306, 308, 310, 312 and 314 in FIG. 3. A peak areadeterminer 404 determines an area of each identified peak based on apre-defined width of the peaks. The pre-defined width may correspond tothe entire area of each peak or a sub-portion thereof, such as a widthat half the height of the peaks. Using a wider width may increase thesignal-to-noise ratio. FIG. 5 shows a magnified view of the peaks 306,308, 310, 312 and 314 of FIG. 3, an example range 502 for determiningpeak area, and an axis 503 extending through a maximum height of thepeaks.

Returning to FIG. 4, a ratio determiner 406 determines ratios of theareas of the peaks 308-314 to the area of the peak 306, whichcorresponds to the detector channel for the dye 230. A color bleed (CB)factor determiner 408 determines a set of color bleed factors for thedetector channels based on the ratios. For the initial set of colorbleed factors, this may performed during a factory calibration beforethe apparatus 102 is used to process samples. A color bleed (CB) factorcorrector 410 determines a correction for color bleed factors, whichwere previously determined by the color bleed factor determiner 408and/or otherwise, due to any detector channel gain changes over time.The correction determination may be performed before, concurrently withand/or after processing a sample.

As briefly discussed above, the color bleed factor determiner and/orcorrector 116 can additionally or alternatively determine the correctionto the color bleed factors based on one or more gain-monitoring fixture118. FIG. 6 shows an example of the one or more gain-monitoring fixture118 in connection with a sub-portion of the sample processing apparatus102.

In the illustrated embodiment, the sample carrier 104 is located in thesample carrier receptacle 106, and the sample carrier 104 includessample channels 606 ₁, 606 ₂, 606 ₃, . . . , and 606 _(N) with separatedfragments 608 ₁, 608 ₂, 608 ₃, . . . , and 608 _(N) positioned atreading regions 610 ₁, 610 ₂, 610 ₃, . . . , and 610 _(N) located alonga reading path 612. The optical reader 110 (FIG. 1) sequentiallyprocesses (i.e., illuminates and detects emissions from) the fragments608 at the reading regions 610.

In the illustrated embodiment, gain-monitoring fixtures 118 ₁ and 118 ₂include rigid, transparent, and stabile material such as a plastic orthe like that emits wavelengths in a predetermined range in response tobeing illuminated by the optical reader 110. In one instance, thegain-monitoring fixtures 118 ₁ and 118 ₂ include one or more pieces offluorescent plastic implanted or otherwise affixed to the one or moregain-monitoring fixtures 118.

The gain-monitoring fixture 118 ₁ is located adjacent to the readingregion 610 ₁ and on the reading path 612 and the gain-monitoring fixture118 ₂ is located adjacent to the reading region 608 _(N) and on thereading path 612. With this configuration, the optical reader 110 canprocesses the gain-monitoring fixture 118 ₁ (or 118 ₂), then,sequentially, the fragments 608 at the reading regions 610, and then theother gain-monitoring fixture 118 ₂ (or 118 ₁).

In another embodiment, the one or more gain-monitoring fixtures 118 ₁and 118 ₂ may additionally or alternatively be processed betweenprocessing the fragments 608 at the reading regions 610. In yet anotherembodiment, the one or more gain-monitoring fixtures 118 mayadditionally or alternatively be processed before and/or afterprocessing any of the fragments 608 at the reading regions 610 or any ofthe samples carried by the sample carrier 104.

In another embodiment, the one or more gain-monitoring fixtures 118 areomitted and/or included as part of the sample carrier 104. In anotherembodiment, an additional gain-monitoring fixture(s) 118 is included,for example, as part of the sample carrier (e.g., between channels)and/or as part of the sample processing apparatus 102.

Additionally or alternatively, the sample processing apparatus 102includes one or more gain-monitoring light sources that emit a spectrumcovering a predetermined spectrum such as the emission spectrum of thedyes. The gain-monitoring light source is located in the apparatus 102such that the signal emitted therefrom is detected by the optical reader110, and does not have to be by the sample carrier or along the path612.

Similar to the gain monitoring fixtures 118 ₁ and 118 ₂, thegain-monitoring light source can be used to determine initial andsubsequent detector gains used to determine the gain correction factor.An example of a gain-monitoring light source is a wide-spectrum lightemitting diode (LED) that is switched on and off. Another example ofsuch a gain-monitoring light source includes a plurality of differentcolor LEDs covering the emission spectra of the dyes concurrently emit.

FIG. 7 illustrates an example color bleed factor generator and/orcorrector 116 for correcting color bleed factors based on emissions fromthe one or more gain-monitoring fixtures 118, or gain-monitoring lightsources, detected by the reader 110.

A detector gain determiner 702 determines a gain of each of the detectorchannels of the optical reader 110. In general, the amplitude of eachacquired signal is proportional to the gain of the respective detectionchannel, after subtracting an offset. If the gain is treated as constantfor the run, then the gain can be calculated as the average amplitude ofthe signal. If the signal is considered to vary with time during therun, then the gain can be fit to a polynomial function.

A reference detector identifier 704 identifies one of the detectors as areference detector. A gain ratio determiner 706 determines a ratio ofthe gain of each of the detectors to the gain of the reference detector.A gain factor determiner 708 determines an initial gain factor for thedetection channels based on the ratios. These ratios may be saved asreference gain ratios during factory calibration of the apparatus 102.

A gain factor corrector 710 corrects a previously determined detectorchannel gain for relative detector channel gain changes over time. Inthe illustrated embodiment, this includes utilizing ratios subsequentlydetermined before, during and/or after processing a sample, andadjusting one or more of the color bleed factors, output of the opticalreader 110 before color separation or the color separated signal basedon a change between the subsequently determined ratios and the referencegain factor.

FIG. 8 illustrates a method for determining and/or correctingcolor-bleed factors based on a calibration substance.

At 802, a sample carrier carrying a color bleed calibration substance isloaded in the apparatus 102. As described herein, a suitable color bleedcalibration substance includes a plurality of different size fragmentsin which the fragments are divided into multiple groups to be labeledwith a distinct dye for each group.

At 804, the calibration substance is processed by the processingstations 108. This includes separating fragments thereof based onfragment size.

At 806, the separated fragments are illuminated with a light source.

At 808, emissions in response to the illumination are detected andprocessed by a plurality of detector channels, each channel beingconfigured to detect a signal corresponding to the ideal non-overlappingspectrum of a different dye.

At 810, each channel generates an output in which the signal amplitudeindicates the amount of the emitted signal detected.

At 812, the signal amplitudes from all the channels are summed for eachacquisition time.

At 814, a maximum height of summed signal is identified, and itindicates the presence and the center of a peak generated from afragment.

At 816, a peak area for each of the individual channel outputs for eachacquisition time is determined based on corresponding identified peaksand a predefined acquisition-time range around the peak center.

At 818, a reference color bleed factor for each of the detectors foreach of the dyes is generated based on a ratio of the peak areas in theoutput of the detectors for a dye to the peak area in the output of thedetector corresponding to the dye.

At 820, a correction factor for the set of color bleed factors isdetermined before, during, and/or after processing a DNA sample.

At 822, the reference color bleed factors are corrected, if needed,based on the correction factor.

At 824, the corrected reference color bleed factors are utilized tocolor separate mixed signals corresponding to processed DNA samples.

FIG. 9 illustrates a method for correcting color-bleed factors based onemission from one or more gain-monitoring fixtures and/or alight-emitting device.

At 902, color bleed factors for each of the detectors for each of thedyes are determined. In one instance, the color bleed factors aredetermined using a calibration substance as described herein.

At 904, reference relative gain ratios are determined for the detectorsusing the one or more gain-monitoring fixtures and/or the light source.

At 906, before, during and/or after an acquisition time, subsequentrelative gain ratios are determined for the detectors using the one ormore gain-monitoring fixtures and/or the light source.

At 908, a gain correction is determined based on the reference relativegain ratios and the subsequently determined relative gain ratios.

At 910, the gain correction is employed to scale one of the color bleedfactors. Alternatively, the gain correction is used to scale the outputsignals of the optical reader 110 before the color separation.

It is to be appreciated that the methods herein can be implemented viaone or more processor of one or more computing systems executing one ormore computer readable and/or executable instructions stored on computerstorage medium such as memory local to or remote from the one or morecomputing systems.

The following describes embodiments herein in mathematical terms.

The fluorescent light intensity from dye i is X_(i) and the lightintensity detected through detector channel j is Y_(j). The acquiredsignal from each detection channel contains substantial amount of offsetand certain amount of background signal. The background signal is mostlythe excitation light scattered by the biochip material surrounding thecapillary. The amount of these offset and background signal are fairlyconstant throughout the data acquisition, and can be calculated and usedfor baseline correction. The variable Y_(j) is the signal amplitudeafter the baseline has been subtracted from the acquired signal.

For an example with five (5) dyes and five (5) detection channels, thedetected signal for channel j can be written as the combination offluorescent light from five (5) dyes as shown in Equation 1:

Y _(j) =A _(j1) *X ₁ +A _(j2) *X ₂ +A _(j3) *X ₃ +A _(j4) *X ₄ +A _(j5)*X ₅.  Equation 1

The coefficient A_(ji) can be considered as the color-bleed factor fromdye i to detection channel j, if i is not the same as j. For the case ofi=j, the coefficient A_(ii) represents the detection efficiency of a dyeby its principle channel. It is the principle coefficient, which has thelargest value.

The color bleed effect can be described through Equation 2:

$\begin{matrix}{Y_{j} = {\sum\limits_{i = 1}^{5}{A_{ji}*X_{i}}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

with j=1, 2, . . . , 5.

If X is the vector of the dye emission intensities, Y is the vector ofthe detected signal amplitudes, and A is the matrix of the color-bleedfactors, the foregoing can be written as a matrix operation as shown inEquation 3:

Y=AX.  Equation 3

It describes the relationship between the dye emission intensity and thedetected signal amplitude in a set of simultaneous equations. Theunknown dye emission intensity X can be solved by using the inversematrix of A, as shown in Equation 4:

B=A⁻¹.  Equation 4

Then, the dye emission intensity X is given by Equation 5:

X=B Y,  Equation 5

and in expanded terms as show in Equation 6:

$\begin{matrix}{X_{i} = {\sum\limits_{j = 1}^{5}{B_{ij}*{Y_{j}.}}}} & {{Equation}\mspace{14mu} 6}\end{matrix}$

While the color-bleed factors A_(ji) are all positive values, theinverse matrix coefficients B_(ij) can be positive and negative values.In one instance, it is positive for the diagonal elements and mostlynegative for the other elements. The calculation for the dye emissionintensities can be considered as de-convolution of the detected signalamplitudes.

If the peak is originated from a fragment attached with dye i, the peakarea is denoted as P_(ji) for the color signal j. The peak area P_(ji)measures the fluorescent light intensity emitted by fragments of samesize with dye i and detected by the detector channel j under certainscale.

According to Equation 1, it is actually measuring the color bleed factorA_(ji). It is different from A_(ji) only by a scaling factor s_(i). Thisscaling factor depends on the amount of fragments attached with dye i.as shown in Equation 7:

P _(ji) =s _(i) *A _(ji).  Equation 7

If there are m different fragment sizes attached with dye i, thevariable k can be used as the index of the fragment size, with k rangefrom 1 to m, rendering Equation 8:

P _(ji)(k)=s _(i)(k)*A _(ji).  Equation 8

The peak areas generated by all fragments in the color signal with dye iand detected by detector channel j are summed to yield Equation 9:

$\begin{matrix}{Q_{ji} = {\sum\limits_{k = 1}^{m}{{P_{ji}(k)}.}}} & {{Equation}\mspace{14mu} 9}\end{matrix}$

The sum of s_(i)(k) can be represented as shown in Equation 10:

$\begin{matrix}{R_{i} = {\sum\limits_{k = 1}^{m}{{s_{i}(k)}.}}} & {{Equation}\mspace{14mu} 10}\end{matrix}$

Combining the above equations, renders Equation 11:

Q _(ji) =R _(i) *A _(ji).  Equation 11

Now, based on above equation, the ratio of the coefficient A_(ji)against the principle coefficient A_(ii) can be calculated as shown inEquation 12:

A _(ji) /A _(ii) =Q _(ji) /Q _(ii).  Equation 12

The above equation is applicable for indices i and j. A_(ii) representsthe fraction of the fluorescent light from dye i that is detected by thedetector channels i. It depends on the emission spectrum of dye i aswell as the filtering spectrum of the detector channel i. It can becalculated from the theoretical basis or measured.

A preferred procedure is to start with an estimated fraction (less thanone) as the basis to calculate the other coefficients A_(ji) accordingto above equation. These preliminary values are then used to normalizethem such that the sum of these coefficients becomes 1. Let thenormalized principle coefficient be c_(i), the color-bleed factors canbe expressed as shown in Equation 13:

A _(ji) =c _(i)*(Q _(ji) /Q _(ii)) with i,j=1, 2, 3, 4, 5, . . .  Equation 13

In summary, based on the signals acquired from the color-calibrationsubstance, the peak areas of the known fragments for each dye i arecalculated and summed for each color signals j as Q_(ji). From thesevalues of Q_(ji), the color-bleed factors, or a matrix coefficientsA_(ji), can be calculated using Equation 13.

The color-bleed factors may depend on the position of the excitationspot. As such, they can be represented as a function of position alongan excitation axis λ as A_(ji)(λ). At the center spot position, λ=0, andthe color-bleed factors become A_(ji)(0). The position dependentcolor-bleed factors can be written as shown in Equation 14:

A _(ji)(λ)=(1+δ_(j)(λ))A _(ji)(0) with i,j=1, 2, 3, 4, 5, . . .  Equation 14

In this equation, the term δ_(j)(λ) is the position-dependent correctionfactor. It is a function to be determined by fitting the calculatedA_(ji)(λ) values to a polynomial function at multiple λ positions.Assume the polynomial is a quadratic function, then it needs at leastthree A_(ji)(λ) values at three different λ positions.

Suppose the color calibration substance is running through Xcapillaries, at the same time or at different times, at positions λ₁,λ₂, . . . , λ₉. Firstly, the color-bleed factors from N dyes can beaveraged to enhance the accuracy as shown in Equation 15:

$\begin{matrix}{{{f_{j}\left( \lambda_{k} \right)} = {{\sum\limits_{i = 1}^{5}{{{A_{ji}\left( \lambda_{k} \right)}/5}\mspace{14mu} {with}\mspace{14mu} k}} = 1}},2,\ldots} & {{Equation}\mspace{14mu} 15}\end{matrix}$

The f_(j)(λ_(k)) values are then used to fit the quadratic function ofEquation 16:

g _(j)(λ)=a _(j0) +a _(j1) λ+a _(j2)λ².  Equation 16

The fitting results, a_(j0), a_(j1), and a_(j2), are the quadraticfunction coefficients for the term (1+δ_(j)(λ)) in Equation 14. They areused to calculate for the color-bleed factors A_(ji)(λ) according toEquation 14 as shown in Equations 17 and 18:

A _(ji)(0)=a _(j0),  Equation 17

and

δ_(j)(λ)=(a _(j1) /a _(j0))λ+(a _(j2) /a _(j0))λ².  Equation 18

In addition to the dependence of the excitation position, thecolor-bleed factors may also vary with the DNA locus of the fragment.This should not occur for an ideal dye. However, the dye is attached toa primer, and the primer is specific for each locus. The chemicalenvironment of the primer may affect the fluorescent spectrum of certaindyes. If the color-bleed factors for a locus differ significantly fromthat of other loci, then it is desirable to calculate the color-bleedfactors specifically for that locus. This can be done by simplyselecting and summing the peak areas only the fragments within the sizerange of the locus, and use them to calculate Q_(ji) as shown inEquation 9. In this case, there are special sets of color-bleed factors,A_(ji)(λ), prepared for these special loci. Each special set is used,according to the same Equation 6, for color separation of the signalswithin the fragment size range of the special locus.

The color calibration substance can be prepared in different forms. Forexample, it may contain fragments of only one dye and it is used todetermine the color-bleed factors of this dye. In this way, it can havemore fragments of this dye in the substance. However, another substanceis needed to prepare and calculate the color-bleed factors for anotherdye. Likewise, one substance may contain fragments of two dyes andanother substance contains fragments of other dyes. In a calibrationprocedure, these multiple substances can run simultaneously in separatecapillaries of the same biochip. However, for a routine DNA analysis,multiple substances for color calibration take away the sample space andwhich may not be desirable.

With respect to detector gain, the color-bleed factor A_(ji) can beseparated into two terms representing the two stages of the color-bleedprocess as shown in Equation 19:

A _(ji) =G _(j) *α _(ji).  Equation 19

The first term α_(ji) describes the combined effect of fluorescentspectrum and the filter characteristics. The second term, G_(j),describes the optical detection and amplification. It can be consideredas the gain of the detection channel. In terms of gain, color bleed canbe represented as shown in Equation 20:

$\begin{matrix}{Y_{j} = {G_{j}*{\sum\limits_{i = 1}^{5}{\alpha_{ji}X_{i}}}}} & {{Equation}\mspace{14mu} 20}\end{matrix}$

with j=1, 2, . . . .

Due to variation of the gain over time, the gain changes to G′_(j) atthe run time, and the detected signal represented as shown in Equation21:

$\begin{matrix}{Y_{j} = {G_{j}^{\prime}*{\sum\limits_{i = 1}^{5}{\alpha_{ji}{X_{i}.}}}}} & {{Equation}\mspace{14mu} 21}\end{matrix}$

If the gain G′_(j) is measured for each detection channel, it can beused to modify the color-bleed factors as shown in Equation 22:

A′ _(ji) =G′ _(j)*α_(ji)=(G′ _(j) /G _(j))*A _(ji).  Equation 22

The modified matrix A′ is then used to find the inverse matrix B, andthe color-separated signal X can be calculated according to Equation 6.

This can be alternatively be expressed as shown in Equation 23:

$\begin{matrix}{{\left( {G_{j}/G_{j}^{\prime}} \right)*Y_{j}} = {\sum\limits_{i = 1}^{5}{A_{ji}{X_{i}.}}}} & {{Equation}\mspace{14mu} 23}\end{matrix}$

The detected signal Y_(j) is scaled as show in Equation 24:

Y′ _(j)=(G _(j) /G′ _(j))*Y _(j).  Equation 24

Follow Equations 5 and 6, the unknown X_(i) can be calculated from theoriginal color-bleed factors A_(ji) as shown in Equation 25:

$\begin{matrix}{X_{i} = {\sum\limits_{i = 1}^{5}{B_{ij}{Y_{j}^{\prime}.}}}} & {{Equation}\mspace{14mu} 25}\end{matrix}$

Using the relative gain r₁ for channel j, with respect to the gain of areference channel, the ratio of the gains can be represented as shown inEquations 26 and 27:

r _(j) =G _(j) /G ₁, and  Equation 26

r′ _(j) =G′ _(j) /G′ ₁.  Equation 27

Using Equations 26 and 27, Equation 23 can be written as shown inEquation 28L

$\begin{matrix}{{\left( {r_{j}/r_{j}^{\prime}} \right)*Y_{j}} = {\left( {G_{1}^{\prime}/G_{1}} \right)*{\sum\limits_{i = 1}^{5}{A_{ji}{X_{i}.}}}}} & {{Equation}\mspace{14mu} 28}\end{matrix}$

The detected signal is first scaled by the relative gain as shown inEquation 29:

Y′ _(j)=(r _(j) /r′ _(j))*Y _(j).  Equation 29

X′_(i) can be represented as show in Equation 30:

X′ _(i)=(G′ ₁ /G ₁)*X _(i).  Equation 30

Using Equations 5 and 6, and the original color-bleed factors A_(ji),renders Equation 31:

$\begin{matrix}{X_{i}^{\prime} = {\sum\limits_{i = 1}^{5}{B_{ij}{Y_{j}^{\prime}.}}}} & {{Equation}\mspace{14mu} 31}\end{matrix}$

The unknown X_(i) can be calculated as shown in Equation 32:

X _(i)=(G ₁ /G′ ₁)*X′ _(i).  Equation 32

This is just a constant scaling of a factor close to one for all colorseparated signals. It can be omitted, in which we just use X′_(i) forX_(i). If it is desirable to take account of this constant scaling, thenit is preferred to scale the detected signal as shown in Equation 33:

Y′ _(j)=(G ₁ /G′ ₁)*(r _(j) /r′ _(j))*Y _(j).  Equation 33

The unknown X_(i) is then given by the result of the inverse matrixB_(ij) multiplied by Y′_(j).

In summary, in the calibration for the color-bleed factors A_(ji), theratio of the gain for every detection channel with respect to areference channel is calculated and stored as r_(j). During a run withDNA samples, the gains of all channels are measured and the relativegain r′_(j) is calculated for every detection channel with respect tothe same reference channel as in the calibration. These relative gainsare used to scale detected signal before the color separation using theoriginal color-bleed factors A_(ji). The ratio of the reference channelgains, G₁/G′₁, can be used to scale the detected signal.

The application has been described with reference to variousembodiments. Modifications and alterations will occur to others uponreading the application. It is intended that the invention be construedas including all such modifications and alterations, including insofaras they come within the scope of the appended claims and the equivalentsthereof.

1. A method, comprising: calibrating color bleed factors of opticaldetector channels of a sample processing apparatus through processing acolor bleed calibration substance which includes a plurality ofdifferent size fragments replicated from different groups of DNA loci,wherein fragments in a same group are labeled with a same fluorescentdye, and fragments in different groups are labeled with differentfluorescent dyes having different emission spectra, wherein thedifferent size fragments are processed during different acquisitiontimes.
 2. The method of claim 1, wherein the optical detector channelsdetect a fractional amount of signal emitted from a fluorescent dye thatis illuminated with a light source.
 3. The method of claim 2, whereinthe fractional amount represents the color bleed factors of the opticaldetector channels for the dye.
 4. The method of claim 2, wherein outputsignals of the optical detector channels respectively include peakamplitudes indicative of the fractional amounts.
 5. The method of claim4, further comprising: determining a common center and individual areasfor the identified peak; determining a maximum height of the amplitudesummed at each acquisition time; determining a center of the peak basedon the maximum height; determining a peak area for each optical channelbased on a corresponding identified peak and a predefined acquisitiontime range around the corresponding peak center.
 6. The method of claim5, further comprising: determining a total area for each detectorchannel; and determining ratios of the total area of each detectorchannel to the total area of the detector channel corresponding to theemission spectrum of the emitted signal.
 7. The method of claim 6,further comprising: calculating color-bleed factors within a particularlocus by determining a total peak area based only on fragments within afragment size range corresponding to the locus.
 8. The method of claim6, further comprising: calculating multiple sets of color-bleed factorsin which each set includes only fragment peaks located within a certainrange of fragment sizes, and the set is used for color separation of thesignals acquired within that section of fragment size.
 9. The method ofclaim 5, further comprising: generating, subsequently, a color bleedfactor correction for the optical detector channels using the colorbleed calibration substance at least one of before, during or afterprocessing a DNA sample.
 10. The method of claim 9, further comprising:correcting the set of relative color bleed factors based on the colorbleed factor correction, wherein correcting includes one of scaling therelative color bleed factors or correcting the relative color bleedfactors for changes in gain of the optical detectors channels.
 11. Themethod of claim 10, further comprising: color separating an outputsignal, of the optical detector channels, indicative of processed DNAfragments using the corrected color bleed factors.
 12. The method ofclaim 1, wherein the color bleed calibration substance is included inone or more channels of a sample carrier inserted in and processed bythe sample processing apparatus and is processed at least one of beforeor after processing a DNA sample with the sample processing apparatus.13. The method of claim 1, wherein the color bleed calibration substanceis included in one or more channels of a sample carrier inserted in andprocessed by the sample processing apparatus, wherein the sample carrierconcurrently carriers at least one DNA sample in at least one otherchannel, and the color bleed calibration substance and the at least oneDNA sample are concurrently processed.
 14. The method of claim 1,wherein at least one of the color bleed calibration substance is apositive control sample or a positive control sample is used as thecolor bleed calibration substance.
 15. A method, comprising: generatinga first signal indicative of a reference gain of optical detectors of asample processing apparatus based on a first emission from again-monitoring material of the sample processing apparatus in responseto illuminating the gain-monitoring material; generating a second signalindicative of a subsequent gain of the optical detectors based on asecond emission from a gain-monitoring material in response toilluminating the gain-monitoring material; and scaling at least one ofcolor bleed factors of the sample processing apparatus or data acquiredby the sample processing apparatus based on a signal indicative of adifference between the reference gain and the subsequent gain.
 16. Themethod of claim 15, wherein the second signal is determined at least oneof before, during or after an acquisition time in which one or more DNAsamples are processed by the sample processing.
 17. The method of claim15, wherein the gain-monitoring material is part of the sampleprocessing apparatus.
 18. The method of claim 15, wherein thegain-monitoring material is part of a sample carrier received by thesample processing apparatus, wherein the sample carriers includes one ormore channels for carrying samples to be processed by the sampleprocessing apparatus.
 19. The method of claim 15, wherein thegain-monitoring material includes one or more fluorescent materials thathave a known emission spectrum within a predetermined emission spectrumof interest.
 20. The method of claim 17, wherein the gain-monitoringmaterial is illuminated by a source of the sample processing apparatusused to illuminates separated DNA fragments.
 21. The method of claim 17,wherein the gain-monitoring material is illuminated by a source of thesample processing apparatus that is different from a sample source usedto illuminates separated DNA fragments.
 22. A sample processing system,comprising: a sample carrier receptacle configured to receive a samplecarrier carrying one or more samples to be processed by the sampleprocessing system; one or more processing stations for processing theone or more samples; a reader, including an illumination source and oneor more optical detector channels, that evaluates separated fragments ofa processed sample based on emission spectrums of dyes attached to thefragments, and that generates an output signal; a color separator thatcolor separates a reader output signal corresponding to a processed DNAsample based on color bleed factors of the one or more optical detectorchannels; and a color bleed factor generator and/or corrector configuredto determine color bleed factors for the optical detector channels basedon processing a color bleed calibration substance, wherein the colorbleed calibration substance includes a plurality of different sizefragments in which different size fragments are grouped and labeled withdifferent dye having different emission spectrums in different groups,and the different size fragments are processed and detected overdifferent acquisition times.
 23. The sample processing system of claim22, wherein the color bleed factor generator and/or corrector generatesa reference set of color bleed factors and generates a subsequent colorbleed factor correction at least one of before, during, or afterprocessing the DNA sample, and the bleed factor generator and/orcorrector corrects the reference set of color bleed factors based on thecolor bleed factor correction.
 24. The sample processing system of claim23, wherein the correction corresponds to a change in gain of theoptical detector channels.
 25. The sample processing system of claim 21,wherein the color bleed calibration substance is processed separate fromthe DNA samples.
 26. The sample processing system of claim 21, whereinthe color bleed calibration substance is concurrently processed with theDNA sample.
 27. A sample processing system, comprising: a sample carrierreceptacle configured to receive a sample carrier carrying one or moresamples to be processed by the sample processing system; one or moreprocessing stations for processing the one or more samples; again-monitoring material that emits fluorescent light with a widespectrum. a reader, including an illumination source and one or moreoptical detector channels, that evaluates the gain-monitoring materialand separated fragments of a processed sample based on emissionspectrums of dyes attached to the fragments, and that generates anoutput signal; a color separator that color separates an output signalof the reader corresponding to a processed DNA sample based on colorbleed factors of the one or more optical detector channels; and a colorbleed factor generator and/or corrector configured to determine a gainof the detector channels based on the signal emitted by the gain monitormaterial and correct the color bleed factors for changes in gain of theoptical detector channels.
 28. The system of claim 27, wherein the colorbleed factor generator and/or corrector generates a reference gain and asubsequent gain at least one of before, during, or after processing theDNA sample, and the color bleed factor generator and/or correctorcorrects for changes in gain of the optical detector channels based on adifference between the reference and subsequent gains.
 29. The system ofclaim 27, wherein the gain-monitoring material is part of the sampleprocessing apparatus.
 30. The system of claim 27, wherein thegain-monitoring material is part of the sample carrier.